Nuclear receptors (NRs) modulate transcription through interaction with co-activators and co-repressors. A wide body of research has been carried out with the DRIP/TRAPs and the p160/SRC family of co-activators, and with SMRT/N-CoR co-repressors. During the last granting period, our laboratory cloned and characterized four novel factors (NRC, NIF-1, NRIF3, and PSF-A) that appear to play an important role in mediating the activity of NRs and other transcription factors. NRC (Nuclear Receptor Co-regulation) is a novel 2063 amino acid nuclear protein with two LxxLL motifs. LxxLL-1 interacts with all NRs while LxxLL-2 interacts with ERbeta and LXRbeta. The binding of liganded-receptor with LxxLL-1 results in a conformational change in NRC leading to transcriptional activation. NRC interacts with CBP in vivo and also activates other transcription factors such as cFos and cJun. NIF-1 (NRC Interacting Factor-I) is a novel 1,342 amino acid nuclear protein that interacts in vivo and in vitro with NRC and enhances ligand-dependent transcriptional activation by NRs and cFos and cJun through association with NRC. NRIF3 (Nuclear Receptor Interacting Factor 3) is a 177 amino acid nuclear protein that only interacts with TRs and RXRs and contains both activation and repression domains. Repression is dependent on phosphorylation of Ser28. Thus, NRIF3 could act as an activator or repressor depending on the state of phosphorylation. In a number of breast cancer cells, NRIF3 is not an activator and its expression rapidly leads to caspase-2 mediated apoptosis. PSF-A is a 707 amino acid nuclear protein that associates with the DBD of many NRs and acts as a repressor through its association with Sin3 and HDAC1 and HDAC2. The focus of this proposal is to identify novel factors, through yeast two-hybrid screens and mass spectroscopy, that complex with and modulate the activity of NRC, NIF-1, and NRIF3. We also plan to further clarify the role of PSF on modulating gene expression by a variety of NRs. We developed NRIF3 -/- mice to study the physiological role of NRIF3. Although NRC +/- mice exhibit embryonic lethality, and NRC +/- mice are normal, we developed a strain of NRC +/- mice that exhibit interesting phenotypes related to NRC haploid deficiency. Identification of factors that associate with these regulators and study of these "knockout mice", should provide a comprehensive picture of how these factors modulate receptor function and other important biological processes in cells.